This invention is in the field of spectrophotometric determinations of concentrations of substances in solution and relates to methods of calibration of apparatus used to measure quantity of one or more analytes or interferents in a serum or plasma specimen, using synthetic fluids.
Clinical laboratory tests are routinely performed on serum or plasma of whole blood. In a routine assay, red blood cells are separated from plasma by centrifugation, or red blood cells and various plasma proteins are separated from serum by clotting prior to centrifugation. Hemoglobin (Hb), light-scattering substances like lipid particles, and bile pigments bilirubin (BR) and biliverdin (BV) are typical substances which will interfere with and affect spectrophotometric and other blood analytical measurements. Such substances are referred to as interferents, and they can be measured spectrophotometrically. The presence of such interferents affects the ability to perform tests on the serum or plasma and as such can be said to compromise specimen integrity. An apparatus or instrument used for measuring interferents in serum and plasma i.e., assess specimen integrity, is a substitute for visual inspection. The interferents may be regarded as analytes, with respect to the apparatus used to measure the interferents. Because quantitative results from the determination of the concentration of such interferents are reported based on specific calibration algorithms, there is a need to calibrate and to monitor calibration performance daily.
Unlike many blood analytical apparatus, calibration of spectrophotometric apparatus used to measure quantity of one or more analytes or interferents in a serum or plasma specimen is a cumbersome time intensive exercise (Primary Calibration). Each apparatus used for the purposes of determining the concentration of interferents must be calibrated according to this process and over the lifetime of an apparatus can amount to a considerable amount of time and cost. Accordingly, a faster, more cost effective method of calibration is desirable. An example of a method developed to address such problems using synthetic fluids follows: calibrators or synthetic fluids are tested on first instrument that is shown to be in control by quality control fluids. The absorbance measurements are saved. Primary Calibration Algorithms are applied to absorbance measurements made on first instrument, and predict analyte concentration(s). Concentrations of the analyte(s) predicted for calibrators, are stored on a calibration diskette. Install Primary Calibration Algorithms on second instrument.
Test calibrators on second instrument during calibration procedures. Apply Primary Calibration Algorithms to absorbance measurements made on second instrument, and predict analyte concentration(s). For each analyte, plot concentrations from first instrument on the x-axis, against concentrations from second instrument on y-axis, and obtain linear regression equation.
The linear regression equation for a particular analyte would be in the form, y=mx+c, where m is the slope and c is the y-intercept. All subsequent predictions by second instrument for the same analyte would be adjusted by subtracting xe2x80x9ccxe2x80x9d and dividing the difference by xe2x80x9cm.xe2x80x9d
The major disadvantage with this method is that the calibrators need to be carefully designed to produce absorbance spectra that closely resemble the absorbance spectra of serum containing mixed or multiple interferents in the spectral section of the wavelengths used in the calibration algorithm. This can be accomplished through trial and error, but, the entire design and trial-and-error process must be repeated when new calibration algorithms use a different set of wavelengths. Also, such methods are only suitable when the calibration algorithms use a small number of wavelengths.
The present inventor has found that using synthetic fluids it is possible to rapidly and efficiently calibrate an apparatus and use the calibration algorithms of a First Apparatus which is conducted by the standard cumbersome time intensive exercise (xe2x80x9cPrimary Calibrationxe2x80x9d) and a method for calibrating an apparatus based on the calibration of a First Apparatus, and for recalibrating such apparatus, including recalibration of the First Apparatus. According to one embodiment, these apparatus use absorption of radiation to measure analytes in serum or plasma samples. Throughout this application reference is made to measurements through absorption of radiation.
Accordingly, the present invention provides a method for transferring a calibration algorithm from a First Apparatus to a Second Apparatus whereby the Second Instrument need not be calibrated in the same way in which the First Apparatus was calibrated, i.e, by conducting a Primary Calibration.
According to one embodiment the present invention provides a method for transferring a calibration algorithm from a First Apparatus to a Second Apparatus comprising:
(i) conducting a Primary Calibration of the First Apparatus to obtain at least one Primary Calibration Algorithm using a standard set of wavelengths;
(ii) obtaining measurements of absorbance of a set of calibrators on the First Apparatus at the standard set of wavelengths
(iii) obtaining calibration absorbance measurements of the set of calibrators on a Second Apparatus for at least one wavelength from the standard set of wavelengths;
(iv) establishing a First linear regression equation for each wavelength from the standard set of wavelengths using the calibration absorbance measurements from the First Apparatus and the Second Apparatus; and
(v) incorporating the at least one Primary Calibration Algorithm on the Second Apparatus.
According to a method of the invention there is further provided a method of determining the concentration of an analyte in a sample in a Second Apparatus comprising:
(i) transferring a calibration algorithm from a First Apparatus according to the method just outlined above;
(ii) in the Second Apparatus measuring the absorbance of the sample at the standard set of wavelengths;
(iii) adjusting the measurements of absorbance from the sample with the First linear regression equation to obtain a corrected absorbance; and
(iv) calculating a concentration for the analyte using the corrected absorbance. According to a preferred embodiment in the Primary Calibration Algorithms and the calibration absorbance measurements on the First Apparatus are electronically stored and installed on the Second Apparatus, more preferably on a floppy diskette or an EPROM.
According to another embodiment of the invention the samples used for the Primary Calibration are in a first type of vessel and the calibrators are in the same type of vessel, preferably a pipette tip, test tube (labelled or unlabelled), or blood bag tubing.
According to another embodiment of the method four calibrators are used, preferably all from the same batch, most preferably the calibrators mimic hemoglobin, bilirubin, turbidity or biliverdin.
According to another embodiment of the method the calibrators used in the Second Apparatus are exactly the same calibrators used to provide the absorbance measurement on the First Apparatus.
According to another embodiment of the method the standard wavelengths are measured in the near infrared and adjacent visible light spectrum, preferably ten absorbance measurements are taken at each wavelength of the standard wavelength set.
According to yet another embodiment, the method comprises conducting a Primary Calibration of a First Apparatus and using a quality control fluid to confirm the First Apparatus is functioning wherein the quality control fluid, or calibrator, is contained in a sample container. Next, two or more calibrators, called a xe2x80x9ccalibration setxe2x80x9d, are submitted to the First Apparatus and the absorbance for each of the calibrators is measured on the First Apparatus. These absorbance measurements are stored and provide xe2x80x9ccalibration absorbance measurementsxe2x80x9d from the First Apparatus for a calibration set. Also stored are the calibration algorithms from the First Apparatus. Preferably the calibration absorbance measurements and calibration algorithms from the First Apparatus is stored electronically, most preferably on a computer diskette. The calibration algorithms from the First Apparatus are installed in a Second Apparatus, preferably before the Second Apparatus is used. Using similar calibrators from the same batch, i.e., with identical lot number or batch number, which were measured on the First Apparatus and the same xe2x80x9cstandard set of wavelengthsxe2x80x9d, the absorptions of the calibrators are measured on the Second Apparatus to generate a second set of measurements. These absorbance measurements from the Second Apparatus are also stored with the absorbance measurements from the First Apparatus. Next a first linear regression plot is prepared using the absorbances measured from each of the First and Second Apparatus for each wavelength. Measurements from the First Apparatus are plotted on one axis and measurements from the Second Apparatus are plotted on the second axis; preferably measurements from the First Instrument are plotted on the x-axis. The slope and intercept of this first linear regression plot are stored in the Second Apparatus. Using this stored regression data, the absorbances of actual samples which are measured on the Second Apparatus are adjusted using the stored regression plot for each wavelength. Accordingly, the measurement made in respect of the sample once adjusted, provides a result comparable to that which would be obtained if the sample had been measured on the First Apparatus. In this way, the Second Apparatus need not be calibrated through a Primary Calibration as was done for the First Apparatus.
Accordingly, the present invention provides a method of determining the concentration of an interferent in a sample in a Second Apparatus using a calibration algorithm from a First Apparatus comprising:
(i) conducting a Primary Calibration of the First Apparatus to obtain at least one Primary Calibration Algorithm using a standard set of wavelengths in the near infrared and adjacent visible range and storing the at least one Primary Calibration Algorithm on a floppy diskette;
(ii) obtaining calibration absorbance measurements of a set of four calibrators which mimic hemoglobin, bilirubin, turbidity or biliverdin on said First Apparatus at the standard set of wavelengths and storing the calibration absorbance measurements on the floppy diskette;
(iii) installing the calibration absorbance measurements and the at least one Primary Calibration Algorithms stored on the floppy diskette in the Second Apparatus;
(iv) obtaining measurements of absorbance of the set of calibrators on a Second Apparatus for at least one wavelength from the standard set of wavelengths;
(v) establishing a First linear regression equation for each wavelength from the standard set of wavelengths using the calibration absorbance measurements from the First Apparatus and the measurements of absorbance from the Second Apparatus and electronically storing the First linear regression equation in the Second Apparatus;
(vi) in the Second Apparatus measuring the absorbance of the sample at the standard set of wavelengths;
(vii) adjusting the measurements of absorbance from the sample with the First linear regression equation to obtain a corrected absorbance; and
(viii) calculating a concentration for the interferent using the corrected absorbance.
As will be readily apparent to those skilled in the art, this set of measurements obtained from a First Apparatus, the xe2x80x9ccalibration absorbance measurementsxe2x80x9d, may be applied to any Second Apparatus. Further, the same xe2x80x9ccalibration absorbance measurementsxe2x80x9d may be applied to the First Apparatus in order to recalibrate it. Accordingly, the present invention also provides a method for recalibrating an apparatus where the apparatus has the calibration algorithm from the First Apparatus installed. As is readily apparent, the calibration set may also be used to recalibrate any Second Apparatus so requiring recalibration.
According to an embodiment of this aspect of the invention, the calibrators are measured in the apparatus which is being recalibrated and absorbances recorded using the same standard set of wavelengths as used in the First Apparatus. New, or Second linear regression plot(s) is (are) then calculated for each wavelength with this set of calibration absorbance measurements versus the measurements obtained from the First Apparatus to generate a Second linear regression plot having an intercept and slope, which is then stored in the apparatus being recalibrated. Measurements of actual samples in the recalibrated apparatus are then adjusted using the Second linear regression plots.
Accordingly, the present invention provides a method for recalibrating a First Apparatus and/or a Second Apparatus comprising:
(i) incorporating in the First and/or Second Apparatus at least one Primary Calibration Algorithm and calibration of absorbance measurements of a set of calibrators at a standard set of wavelengths, obtained from a Primary Calibration on the First Apparatus;
(ii) obtaining measurements of absorbance of the set of calibrators on the First and/or Second Apparatus for at least one wavelength at the standard set of wavelengths; and
(iii) establishing a Second linear regression equation using the incorporated calibration absorbance measurements and the measurements of absorbance obtained on the First and/or Second Apparatus.
As is readily apparent, any such apparatus which has been recalibrated may be used in a method according to the present invention to determine the concentration of an analyte in a sample in an apparatus by steps comprising:
(i) measuring the absorbance of the sample in the recalibrated apparatus at the standard set of wavelengths;
(ii) adjusting the measurements of absorbance from the sample with the Second linear regression equation to obtain a corrected absorbance; and
(iii) calculating a concentration for the analyte using the corrected absorbance. According to a preferred embodiment the samples used for the Primary Calibration are in a first type of vessel and the calibrators are in the same type of vessel, preferably a pipette tip, test tube (labelled or unlabelled), or blood bag tubing.
According to one embodiment of the method of recalibration four calibrators are used, preferably all from the same batch and the calibrators mimic hemoglobin, bilirubin, turbidity or biliverdin.
According to another embodiment of the method the standard wavelengths are measured in the near infrared and adjacent visible light spectrum, preferably ten absorbance measurements are taken at each wavelength of the standard wavelength set.
As stated above, the calibrators used in the methods of the present invention are substances each of which may mimic a constituent in the sample which is being measured by the apparatus, although it is not necessary that they mimic a constituent in the sample.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.